Detection of cell wall galactomannoprotein Afmp1p in culture supernatants of Aspergillus fumigatus and in sera of aspergillosis patients

Mannoproteins are important and abundant structural components of fungal cell walls. The AFMP1 gene encodes a cell wall galactomannoprotein of Aspergillus fumigatus. In the present study, we show that Afmp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmp1p was capable of detecting this protein from the cell culture supernatant of A. fumigatus. The anti-Afmp1p antibody is specific since it fails to react with any protein from lysates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium marneffei, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum by Western blotting. In addition, this Afmp1p antigen-based ELISA is also specific for A. fumigatus since the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that 8 of 15 (53%) patients are Afmp1p antigen test positive. Furthermore, an Afmp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for invasive aspergillosis carry a sensitivity of 86.7% (13 of 15). The specificities of the tests are high since none of the 138 control sera, including 100 from normal blood donors, 20 from patients with penicilliosis marneffei, 6 from patients with candidemia, 8 from patients with typhoid fever, and 4 from patients with melioidosis, was positive by either test. In conclusion, the combined Afmp1p antibody and antigen tests are highly sensitive and specific for A. fumigatus invasive aspergillosis.     

Disseminated penicilliosis marneffei in immunocompetent patients: A report of two cases

Disseminated penicilliosis marneffei is rarely seen in immunocompetent persons. We report here two cases of disseminated penicilliosis marneffei in immunocompetent hosts. Penicillium marneffei disseminated to the brain in one patient and to the bone marrow in the other patient. Both patients received amphotericin B liposome. The cases illustrate the importance of considering penicilliosis marneffei as causes of systemic infections in immunocompetent patients.     

Progressive disseminated penicilliosis caused by Penicillium marneffei. Report of eight cases and differentiation of the causative organism from Histoplasma capsulatum

Eight patients with fatal penicilliosis caused by Penicillium marneffei are reported. All were natives of southern rural Guangxi, and none had a predisposing illness or evidence of altered immunity. The distinctive features of P. marneffei include proliferation of yeast-like cells within histiocytes, followed by the development of focal necrosis and, eventually, large abscesses. Outside the histiocytes, the fungi elongate, become slightly curved, and form septa. In vitro, P. marneffei produces a red pigment which diffuses into the culture medium. The differentiation between P. marneffei and Histoplasma capsulatum is described, and possible reservoirs for P. marneffei are discussed.     

Detection and characterization of IgE-binding factors (IgE-BF) within supernatants of the cell line RPMI-8866, normal human sera and sera from atopic patients.

IgE-binding factors (IgE-BF) have been shown to be important regulatory factors for IgE induction and suppression. The analysis of IgE-binding factor activity by a modified inhibition radioimmunoassay (RIA), as well as by monoclonal antibodies (mAb) was carried out in the supernatant of the Fc epsilon RII+ cell line RPMI-8866 as well as in human sera. Kinetics of IgE-BF showed optimal release from RPMI-8866 cells after 3-4 days. Gel filtration of the supernatant indicated binding activity at less than 100,000, 45,000 and 25,000 MW. Within normal human sera two peaks of IgE-BF activity were obtained at 45,000 and 25,000 MW. In sera with high IgE levels (atopic dermatitis) a peak at less than 100,000 was MW detected. Within this peak endogenous IgE was present. Addition of sodium dodecylsulphate induced a release of IgE-BF with a MW of 60,000.     

应用蛋白质L检测和纯化培养上清液中的单链抗体

目的 探索一种检测和纯化单链抗体的新方法。方法 使用不同的酶标记物，观察ELISA法检测培养上清液中单链抗体的相对灵敏度。含单链抗体的培养上清液经超滤、313g／L饱和硫酸铵沉淀及蛋白质L琼脂糖亲和层析纯化单链抗体。结果 HRP标记蛋白质L可检测到1：16孔的阳性结果，而HRP标记蛋白质A仅检测到1：2孔的阳性结果。用9E10单抗检测培养上清液中的单链抗体时，除加入未经稀释培养上清液孔略有显色外，其他加入稀释后的培养上清液孔均不显色。蛋白质L亲和层析纯化的单链抗体经SDS-PAGE及ELISA鉴定，所得到的单链抗体纯度高、活性保持良好。结论 蛋白质L是一种能灵敏检测和有效纯化单链抗体的结合物，本实验建立的方法是一种有效、简便实用的方法。     

Unusual pulmonary manifestations of disseminated Penicillium marneffei infection in three AIDS patients.

Penicillium marneffei is a rare fungal pathogen which can cause human infections in people predominantly living in South-east Asia and the southern portion of China. We report three cases of systemic P. marneffei infection in patients infected with HIV who lived in or had travelled to endemic areas. The clinical manifestation includes high fever, chills, weight loss, general malaise, chronic cough, haemoptysis, multiple skin lesions, abnormal liver function, etc. Chest X-ray showed single or multiple cavitary lesions with smooth or irregular thin wall. P. marneffei is cultured from blood, sputum, skin biopsy, sono-guide aspiration and bronchoscopic biopsy. After antifungal therapy with intravenous amphotericin B or oral fluconazole, skin lesions resolved completely within 2 weeks and cavitary lesions in the lungs changed to chronic fibrotic and interstitial processes after several months to a few years later. Our two cases had been treated as either pulmonary tuberculosis or suspected malignancy. A definite diagnosis and early treatment are important because this fungal infection is a marker of AIDS in South-east Asia.     

Detection of antigens with affinity for host cell membrane polypeptides in culture supernatants of Trypanosoma cruzi.

Parasite antigens which bind to host cell molecules of approximately 32 and 34 kilodaltons (kDa) were identified in supernatant fluids obtained from axenic cultures of Trypanosoma cruzi. These parasite components were first detected in culture supernatants obtained after 2 weeks in culture. Immunoblot analysis of culture supernatants exhibiting binding activity revealed the presence of several parasite antigens ranging in molecular mass from approximately 26 to 290 kDa. Gel filtration (Sephacryl S-300) analysis of culture supernatants revealed four major peaks, but only the highest-molecular-mass peak (containing several parasite antigens ranging from 27 to 250 kDa) possessed binding activity for the host cell molecules.     

伏立康唑与两性霉素B脂质体治疗艾滋病合并播散性马尔尼菲青霉菌病的对照研究

目的比较伏立康唑和两性霉素B脂质体治疗艾滋病（AIDS）合并播散性马尔尼菲青霉菌病（PSM）的疗效及安全性。方法对经血/骨髓培养确诊为AIDS合并PSM的患者,分别给予伏立康唑和两性霉素B脂质体治疗,疗程28d,比较两组的疗效和安全性。结果共52例患者纳入研究,其中伏立康唑治疗组20例,两性霉素B脂质体治疗组32例。治疗14d和28d时,伏立康唑组和两性霉素B脂质体组的治疗有效率分别为40.0%、65.0%和56.3%、71.9%,两组比较差异均无统计学意义（Z=1.300,P=0.254;Z=0.273,P=0.601）,但两组28d的治疗有效率均明显高于14d（Z=3.994,P=0.046）。治疗过程中,两组均未出现因毒副作用停药的现象,但两性霉素B脂质体组临床毒副反应较重且出现了血清肌酐的升高。结论伏立康唑与两性霉素B脂质体均为治疗AIDS合并PSM的有效方法,但伏立康唑安全性更好。     

Detection of circulating galactomannan in serum samples for diagnosis of Penicillium marneffei infection and cryptococcosis among patients infected with human immunodeficiency virus

Galactomannan (GM) is a heteropolysaccharide in the cell walls of most Aspergillus and Penicillium species. Cross-reactivity of Cryptococcus neoformans galactoxylomannan in an Aspergillus GM test has also been reported. In this study, we used a Platelia Aspergillus enzyme immunoassay kit (Bio-Rad) to test serum samples obtained from 48 human immunodeficiency virus (HIV)-infected patients (15 with penicilliosis [7 with fungemia alone, 4 with cavitary lung lesions alone, 3 with both fungemia and cavitary lung lesions, and 1 with disseminated disease], 22 with cryptococcosis [11 with fungemia alone, 5 with cavitary lung lesions, 3 with both, and 3 with meningitis alone], and 11 without any invasive fungal infection [control]) for GM levels. None of the patients had aspergillosis or concurrent use of piperacillin-tazobactam or amoxicillin-clavulanate. The median time between diagnosis of fungal infection and collection of serum samples was 0 days for penicilliosis and 1.5 days for cryptococcosis. Of patients with penicilliosis, cryptococcosis, and controls, 73.3%, 13.6%, and 9%, respectively, had GM optical density (OD) indices of >0.5 (P = 0.0001). GM OD indices were higher for penicilliosis (median OD index, 4.419; range, 0.158 to >20) than for cryptococcosis (median, 0.247; range, 0.112 to 3.849) cases (P < 0.001). Patients with fungemic penicilliosis had higher OD indices (median, 10.628; range, 0.401 to >20) than patients with nonfungemic penicilliosis (median, 0.378; range, 0.158 to 4.419) and patients with cryptococcemia (median, 0.231; range, 0.112 to 1.168) (P < 0.001). Of the 15 patients with cavitary lung lesions, those with penicilliosis had higher antigen levels (median OD index, 1.641; range, 0.247 to >20) than those with cryptococcosis (median, 0.227; range, 0.112 to 3.849) (P = 0.011). This study showed that the GM OD index was significantly elevated for HIV patients with penicilliosis. The use of the GM antigen assay may facilitate earlier diagnosis of Penicillium marneffei infection for HIV-infected patients in areas of endemicity.     

The decreased number and function of lymphocytes is associated with Penicillium marneffei infection in HIV-negative patients

Background/purposePenicillium marneffei (P. marneffei) infection, which has been traditionally considered as an indicator of immunosuppression, is one of the most common systemic opportunistic infections in patients with AIDS. Recently, more and more P. marneffei infections have been documented in HIV-negative patients without underlying diseases, which challenges the traditional view that P. marneffei infection is an indicator of immunosuppression. We aimed to evaluate the number and function of lymphocytes in HIV-negative patients with P. marneffei infection.Methods15 HIV-negative P. marneffei-infected patients and 18 healthy controls were recruited and investigated. The number and function of lymphocytes were analyzed by flow cytometry.ResultsMost laboratory tests were within the reference ranges, except for a significant increase in total IgE in P. marneffei-infected patients. Lymphocyte subset analysis showed that the number of CD4⁺ T cells and NK cells was significantly decreased in HIV-negative marneffei-infected patients compared with healthy controls. However, almost half of the marneffei-infected patients still had normal levels of lymphocytes. A further analysis of cell function showed that the activation and proliferation of CD4⁺ T cells, the cytotoxicity of CD8⁺ T cells and NK cells, and the cytokine secretion potential of CD4⁺ T cells and NK cells were all impaired, in comparison with healthy controls.ConclusionsP. marneffei infection has to be regarded as an indicator of immunosuppression. A further investigation of cell function is required in patients with opportunistic infection, as the cell function may be impaired in this condition.     

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppress tumor necrosis factor secretion in vitro

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppressed peripheral blood leukocyte secretion of tumor necrosis factor (TNF). The suppressive activities of all three fluids had similar characteristics: each was heat and acid stable, removed by adsorption on immobilized lectins, and abrogated the stimulatory effect of interferon-gamma. Inhibition of leukocyte TNF secretion was observed only when either serum or conditioned medium was added to leukocytes at the initiation of culture; delaying the addition by 2 h failed to suppress cytokine secretion. Suppression by all fluids was also found to be reversible by washing cells free of suppressive activity. Although serum, tumor, and fibroblast culture supernatants inhibited cytokine secretion, they failed to alter the cytotoxic activity of recombinant human TNF on murine L929 cells. This study suggests that factors which can inhibit TNF secretion are present in human blood and are secreted by both fibroblasts and tumor cells. These suppressive factors may play an important role in the regulation of TNF secretion and cytokine homeostasis.     

MP1 Encodes an Abundant and Highly Antigenic Cell Wall Mannoprotein in the Pathogenic Fungus Penicillium marneffei

We cloned the MP1 gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungus Penicillium marneffei. MP1 is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from Escherichia coli to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of P. marneffei. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity.     

Effects of human immunodeficiency virus sera and macrophage supernatants on mesangial cell proliferation and matrix synthesis.

Patients with human immunodeficiency virus (HIV) infection are prone to the development of focal segmental glomerulosclerosis, a lesion in which increased mesangial cell proliferation and matrix synthesis may play a role. We undertook the present study to determine whether HIV sera may affect mesangial cell proliferation and matrix synthesis either directly or indirectly via effects on macrophage supernatants. Pooled HIV sera was found to significantly enhance (P < 0.01) mesangial cell proliferation in a concentration-related manner. Mesangial cell proliferation was significantly suppressed by two medications commonly utilized in HIV-infected patients, azidothymidine and trimethoprim/sulfamethoxazole, and was not significantly altered by lipopolysaccharide, suggesting that these medications as well as recurrent infection are unlikely to account for the proliferative effect of HIV sera. Supernatants from HIV sera-treated macrophages were found to significantly enhance (P < 0.01) mesangial cell incorporation of [3H]proline, a marker for synthesis of the matrix component collagen, compared to supernatants from control sera-treated macrophages. These results suggest that HIV sera may directly enhance mesangial cell proliferation and may indirectly increase mesangial cell matrix synthesis by altering macrophage secretory products. These effects may play a role in the development of glomerulosclerosis in patients with HIV infection.     

Isotype profiles of anti-gp160 antibodies from HIV-infected patients in plasma and culture supernatants

We studied isotype profiles of anti-HIV antibodies (Ab) in HIV-1-infected African patients with high viral loads and major B cell dysfunction. We focussed on IgG1, IgG3, and IgA as these classes and subclasses tend to support neutralizing functions against HIV. Total IgG1, IgG3 and IgA were detected in the plasma of both HIV-1-infected and HIV-negative African individuals, but there was significantly more IgG3, a rare subclass, in HIV-1-infected patients (P < 0.05). Anti-HIV-gp160 specific antibodies were detected in sera from nearly all HIV-1-infected individuals tested, but not in HIV- individuals: 10/10, 9/10 and 8/10 individuals displayed specific IgG1, IgG3 and IgA, respectively. In the corresponding PBMC cultures carried out in the presence of IL-10 and IL-2, there was specific IgG1 and IgA in 5/10, and 3/10, respectively, but no IgG3 was detected. When HAART-treated European HIV-infected PBMC cultures were tested using the same protocol, specific IgG3 was detected in 4/10 cultures, and was unaffected by the addition of soluble CD40L molecules. The present study thus shows that, despite lymph node disorganization in HIV-infected drug-na?ve Africans, these individuals retain the ability to produce HIV-specific IgG1, IgG3 and IgA. However, the specific mechanisms controlling the selective production of IgG3, probably the most potent subclass and a potential target of immuno-intervention, warrants further investigation.     

支原体培养液中抑制HIV—1 RT活性物质的分子性状

对支原体培养液进行的高效液相层析分析的结果显示：具有ＤＮａｓｅ和／或ＲＮａｓｅ活性的两个主要蛋白峰（峰１分子量为６７×１０３～１００×１０３，峰２分子量为１０×１０３～２５×１０３）显示出明显的ＲＴ抑制活性。这一结果说明支原体培养液的ＲＴ活性抑制作用来自于培养液中有ＤＮａｓｅ活性和ＲＮａｓｅ活性的部分，对抑制ＨＩＶ－１活性可能有一定相关。     

Stimulation of natural cytotoxic activity in mixed cell cultures and by culture supernatants containing interferon

We have previously demonstrated that lymphocyte preparations cultured for 5 days with a number of different mitomycin-C-treated lymphoid cell lines showed increased non-specific (NK-like) cytotoxic activity. We have now examined the activity induced in T- and B-cell-enriched responder populations, prepared by sheep red blood cell (SRBC) rosette separation, and found that increased cytotoxic activity was present mainly in the T-cell-enriched fraction.

To investigate whether soluble factors were involved in the mediation of enhanced cytotoxicity, supernatants from mixed cell cultures were used to pretreat freshly isolated effector cell populations before measurement of cytotoxicity against K562 cells. Effector cells pretreated with supernatants from mixed cell cultures incubated for 2–6 days showed increased cytotoxic activity, the greatest activity being obtained with supernatants from 4 and 5 day co-cultures. Increased activity was observed when effector cells were pretreated for 2, 4 and 18 hr with supernatants from mixed cell cultures, regardless of the nature of the stimulator cell (T- and B-cell-derived lines, K562 and normal allogeneic lymphocytes). Active supernatants were produced by T-cell-enriched, but not B-cell-enriched responder populations.

Supernatants from mixed cell cultures were also examined for the presence of interferon (IFN) and in general, those mediating large increases in cytotoxic activity also contained significant anti-viral activity. The IFN detected was not neutralized by antiserum to IFN-β but was partly neutralized by antiserum to IFN-α. pH 2 treatment also removed part of the activity while a combination of pH 2 and anti-α completely neutralized the activity suggesting the presence of IFN-γ and IFN-α. The candidacy of IFNs as mediators of enhanced cytotoxicity in this system is discussed.

     

JEG-3 cell culture supernatants cause reduced interferon-gamma and interleukin-17 production in mixed-lymphocyte reactions

PROBLEM: Immunoregulatory effects of choriocarcinoma-derived factors on leukocytes have been documented. The present study was designed to investigate the effect of JEG-3 culture supernatants on interferon-gamma (IFN-gamma), interleukin-17 (IL-17) and IL-1beta production in the mixed lymphocyte reactions (MLRs). METHOD OF STUDY: A human choriocarcinoma cell line JEG-3 was used to test the effects of its culture supernatants on the proliferation and cytokine production in the MLRs. The cell proliferation was assessed using the BrdU incorporation and the amounts of cytokines were measured using enzyme-linked immunosorbent assays. RESULTS: The JEG-3 culture supernatants caused significantly reduced IFN-gamma and IL-17 production in the MLRs. However, the supernatants did not influence MLR production of IL-1beta. CONCLUSION: IFN-gamma and IL-17 are mainly produced by activated T cells but IL-1beta is primarily produced by monocytes, thus suggesting that immunoregulatory factors of JEG-3 cells selectively inhibit cytokine production by activated T cells rather than that of the monocytes.